Use of circulating cell biomarkers in the blood for detection and diagnosis of diseases and methods of isolating them

ABSTRACT

A new sensitive cell biomarker of solid tumors and viral infection is identified in blood. This biomarker can be used to determine presence of carcinomas, sarcomas, and viruses, rapid determination of treatment response, early detection of cancer, early detection of cancer recurrence, and may be used to determine therapy.

BACKGROUND Field of the Invention

The present invention generally relates to the discovery andcharacterization of biomarkers in blood and other body fluids that canbe used to screen a subject for the presence of solid tumors, aid inselecting a course of cancer therapy, monitor the efficacy of cancertreatments, and detect and monitor viral infections in a subject, cancerscreening, early detection of cancer recurrence, among other importantgoals. The biomarkers of the invention may be used alone or incombination with circulating tumor cells, free plasma and serum DNAcancer markers, cancer-associated protein markers and other biomarkers.

Related Art

When tumor cells break away from primary solid tumors, they penetrateinto the blood or lymphatic circulation, and ultimately leave the bloodstream and enter other organ or tissue to form metastasis. 90% ofcancer-related deaths are caused by the metastatic process. The mostcommon metastatic sites are the lung, liver, bone and brain. Tumor cellsfound in the circulation are called circulating tumor cells (CTCs). Manyresearch publications and clinical trials show that CTCs have clinicalutility in (i) providing prognostic survival and cancer recurrenceinformation through the enumeration of CTCs in the blood stream, and(ii) providing treatment information through examination of proteinexpression levels, and the occurrence of gene mutations andtranslocations in the CTCs. However, CTCs are not consistentlyassociated with the development and/or presence of cancer in a subject,even in stage IV cancer patients.

There are medical conditions in addition to cancer that can be diagnosedthrough the detection of certain cell types in bodily fluids. Inparticular, cells indicative or characteristic of certain medicalconditions may be larger and/or less flexible than other cells found inselected bodily fluids. Accordingly, by collecting such larger and/orless flexible cells from a sample of a bodily fluid, it may be possibleto diagnose a medical condition based on the cells collected.

The identification and characterization of biomarkers in blood and otherbody fluids that can be used to screen a subject for medical conditionswould provide additional tools to a clinician. The present invention isdirected to this and other important goals.

SUMMARY

The present invention is directed to and discloses a type of cell withunique characteristics that is found in the blood of cancer patientshaving solid tumors, including carcinoma, sarcoma, neuroblastoma andmelanoma. The cell, termed “circulating Cancer AssociatedMacrophage-Like cell” (CAML), is described herein and shown to beassociated with the presence of solid tumors in a patient. Fivemorphologies associated with CAMLs have been characterized and described(Adams, D., et al., Circulating giant macrophages as a potentialbiomarker of solid tumors. PNAS 2014, 111 (9):3514-3519 and WO2013/181532). CAMLs are shown through the data presented herein to haveclinical utility in that they can be used as a biomarker for a varietyof medical applications. CAMLs have been found consistently in theperipheral blood of subjects having stage I to stage IV cancer ofepithelial origin by microfiltration using precision microfilters.Additional CAML morphologies, found in blood of cancer patients, arepresented herein.

Medical applications associated with CAMLs include, but are not limitedto, use of the cell as a biomarker to provide a diagnosis of cancer, inparticular, in the early detection of cancer, in the early detection ofcancer relapse or recurrence, and in the determination of cancermutation. CAMLs can also be used as a biomarker in determiningappropriate courses of therapy; in particular, the cells can be used ina rapid determination of effectiveness of chemotherapy and radiationtherapy treatment response.

CAMLS may be used independently as a cancer marker, or in combinationwith CTCs, cell-free DNA, proteins and other biomarkers to provide amore complete understanding of the patient's disease.

More specifically, and in a first embodiment, the present invention isdirected to methods of screening a subject for cancer, comprisingdetecting CAMLs in a biological sample from a subject. In particularaspects, when CAMLs are detected in the biological sample, the subjectis identified as potentially having a solid tumor, such as carcinoma,sarcoma, neuroblastoma or melanoma, among others. In other aspects, whenCAMLs are detected in the biological sample, the subject is identifiedas having a solid tumor, such as carcinoma, sarcoma, neuroblastoma ormelanoma, among others. The identification of the specific type ofcancer can be performed by standard methods, such as staining for cancermarkers. One of the useful attributes of CAMLs is that they can undergoquenching and re-staining, and therefore a large number of cancer cellmarkers can be assayed using the same sample of cells. Theidentification of the specific type of cancer can therefore also beperformed by staining for cancer markers and then re-staining the samecells for additional markers. In certain aspects, the methodsencompassed by this embodiment also include detecting circulating tumorcells (CTCs) and/or white blood cells (WBCs) bound to CTCs in thebiological sample. In particular aspects of this embodiment, the subjectis a subject suspected of having cancer. In particular aspects of thisembodiment, a treatment decision is made based on the outcome of themethod. In particular aspects of this embodiment, the method furthercomprises administering an anti-cancer therapeutic to the subject whenthe subject is identified as having a solid tumor.

In a second embodiment, the invention is directed to methods fordiagnosing cancer in a subject, comprising detecting CAMLs in abiological sample from a subject, wherein when CAMLs are detected in thebiological sample, the subject is diagnosed with cancer. Theidentification of the specific type of cancer can be performed bystandard methods, such as staining for cancer markers. Theidentification of the specific type of cancer can also be performed bystaining for cancer markers and then re-staining the same cells foradditional markers. In certain aspects, the methods encompassed by thisembodiment also include detecting CTCs and/or WBCs bound to CTCs in thebiological sample, wherein when one or more of CAMLs, CTCs and WBCsbound to CTCs are detected in the biological sample, the subject isdiagnosed with cancer. In particular aspects of this embodiment, atreatment decision is made based on the outcome of the method. Inparticular aspects of this embodiment, the method further comprisesadministering an anti-cancer therapeutic to the subject when the subjectis diagnosed with cancer.

In a third embodiment, the invention is directed to methods fordetecting recurrence of cancer in a subject, comprising detecting CAMLsin a biological sample from a subject previously treated for cancer,wherein when CAMLs are detected in the biological sample, recurrence ofcancer is detected. The identification of the specific type of cancercan be performed by standard methods, such as staining for cancermarkers. The identification of the specific type of cancer can also beperformed by staining for cancer markers and then re-staining the samecells for additional markers. In certain aspects, the methodsencompassed by this embodiment also include detecting CTCs and/or WBCsbound to CTCs in the biological sample, wherein when one or more ofCAMLs, CTCs and WBCs bound to CTCs are detected in the biologicalsample, recurrence of cancer is detected. In particular aspects of thisembodiment, a treatment decision is made based on the outcome of themethod. In particular aspects of this embodiment, the method furthercomprises administering an anti-cancer therapeutic to the subject whenrecurrence of cancer is detected.

In a fourth embodiment, the invention is directed to methods forconfirming a diagnosis of cancer in a subject, comprising detectingCAMLs in a biological sample from a subject diagnosed with cancer,wherein when CAMLs are detected in the biological sample, a diagnosis ofcancer is confirmed in the subject. In certain aspects, the methodsencompassed by this embodiment also include detecting CTCs and/or WBCsbound to CTCs in the biological sample, wherein when one or more ofCAMLs, CTCs and WBCs bound to CTCs are detected in the biologicalsample, a diagnosis of cancer is confirmed in the subject. In particularaspects, the initial cancer diagnosis is via mammography, PSA test,presence of CA125, CT, MRI or PET imaging. In a particular aspect, thesubject is suspected of having cancer. The identification of thespecific type of cancer can be performed by standard methods, such asstaining for cancer markers. The identification of the specific type ofcancer can also be performed by staining for cancer markers and thenre-staining the same cells for additional markers. In particular aspectsof this embodiment, a treatment decision is made based on the outcome ofthe method. In particular aspects of this embodiment, the method furthercomprises administering an anti-cancer therapeutic to the subject whendiagnosis of cancer is confirmed in the subject.

In the first through fourth embodiments of the invention, the detectingof WBCs bound to CTCs is determining the number of WBCs bound to CTCs.When CTCs penetrate into the circulation, immune cells (T-cells, asubtype of white blood cells) can recognize them by binding to the CTCs;the immune cells can also kill the CTCs. Filtration of the blood cancapture white blood cells (WBCs) bound to the CTCs. The CTCs can becomedegraded. The presence and/or number of WBCs bound to CTCs in thebiological sample is this an indication of presence of solid tumor andalso the body's ability to eliminate the solid tumors. In certainaspects, the WBCs bound to CTCs in the biological sample are T cells.

In a fifth embodiment, the invention is directed to methods fordetermining cancer stage in a subject, comprising characterizing CAMLsin a biological sample from a subject having a cancer, wherein selectedcharacteristics of the CAMLs indicate cancer stage in the subject. Theidentification of the specific type of cancer can be performed bystandard methods, such as staining for cancer markers. Theidentification of the specific type of cancer can also be performed bystaining for cancer markers and then re-staining the same cells foradditional markers. In certain aspects, the methods encompassed by thisembodiment also include characterizing CTCs in the biological sample,wherein selected characteristics of the CAMLs and CTCs indicate cancerstage in the subject. In certain aspects, the CAMLs and/or CTCs arecollected from the biological sample prior to characterization. Inparticular aspects of this embodiment, a treatment decision is madebased on the outcome of the method. In particular aspects of thisembodiment, the method further comprises administering an anti-cancertherapeutic to the subject after determining cancer stage in thesubject.

In a sixth embodiment, the invention is directed to methods formonitoring efficacy of a cancer treatment, comprising (a) assaying oneor more selected characteristics of CAMLs in a biological sample from asubject undergoing cancer treatment, and (b) comparing assay values forthe one or more selected characteristics determined in (a) to assayvalues for the same characteristics assayed in a similar biologicalsample from the same subject at one or more time points before, duringor after completion of treatment, wherein a change in one or more assayvalues indicates efficacy of the cancer treatment in the subject. Incertain aspects, the methods encompassed by this embodiment also include(a) assaying one or more selected characteristics of CTCs in thebiological sample, and (b) comparing assay values for the one or moreselected characteristics determined in (a) to assay values for the samecharacteristics assayed in a similar biological sample from the samesubject at one or more time points before, during or after completion oftreatment. In certain aspects, the CAMLs and/or CTCs are collected fromthe biological sample prior to characterization. In particular aspectsof this embodiment, a treatment decision is made based on the outcome ofthe method.

In the fifth and sixth embodiments, the selected characteristics of theCAMLs in the sample are one or more characteristics selected from thegroup consisting of:

(i) number of CAMLs;

(ii) average size of the CAMLs (CAML cell sizes range from about 20micron to about 300 microns in diameter);

(iii) average size of the nuclei of the CAMLs (CAMLs have a largeatypical nucleus having a size of about 14-64 μm in diameter);

(iv) morphological shape of the CAMLs (CAML shapes include spindle,tadpole, round, oblong, two legs, more than two legs, thin legs, oramorphous);

(v) CD14 positive phenotype;

(vi) degree of CD45 expression;

(vii) degree of EpCAM expression;

(viii) degree of vimentin expression;

(ix) degree of PD-L1 expression;

(x) degree of monocytic CD11C marker expression;

(xi) degree of endothelial CD146 marker expression;

(xii) degree of endothelial CD202b marker expression;

(xiii) degree of endothelial CD31 marker expression;

(xiv) location of markers (the location markers appear in CAMLs, e.g.,cytoplasm versus nucleus, can change at the different time points);

(xv) presence of one or more markers associated with the cancer in theCAMLs, wherein the marker is diffused, or associated with vacuolesand/or ingested material (e.g., for epithelial cancer, the markers arecytokeratin 8, 18, and 19); and

(xvi) intensity of marker staining.

In the fifth and sixth embodiments, the selected characteristics of theCTCs in the sample are one or more characteristics selected from thegroup consisting of:

(i) number of CTCs;

(ii) number of WBCs bound to the CTCs;

(iii) status of nucleus;

(iv) degree of cytokeratin 8 expression;

(v) degree of cytokeratin 18 expression;

(vi) degree of cytokeratin 19 expression;

(vii) degree of EpCAM expression;

(viii) degree of vimentin expression;

(ix) degree of PD-L1 expression;

(x) degree of uroplakin expression;

(xi) cytokeratin morphology;

(xii) location of markers (the location markers appear in CTCs, e.g.,cytoplasm versus nucleus, can change at the different time points); and

(xiii) intensity of marker staining.

In the fifth and sixth embodiments, the numbers of CAMLs, CTCs and/orWBCs bound to CTCs are determined simultaneously using a microfilter.Suitable microfilters can have a variety of pore sizes and shapes. Inone aspect, the microfilters have pores ranging in size from about 5microns to about 10 microns in size, and may include round, race-trackshaped, oval, square and rectangular pore shapes. In a preferred aspect,the microfilter has precision pore geometry and uniform poredistribution.

The invention is also directed to methods for isolating CAMLs and/orCTCs from a biological sample and counting the isolated cells using acamera, such as a cell phone camera, or white light microscope, or acamera attached to a white light microscope. Thus, and in a seventhembodiment, the present invention is directed to methods for detectingCAMLs and/or CTCs in a biological sample, comprising obtaining abiological sample from a subject, and detecting CAMLs and/or CTCs in thesample, wherein the detecting is via a camera or a white lightmicroscope, or a camera attached to a white light microscope. In certainaspects, the camera is a cell phone camera. In certain aspects, thewhite light microscope has a magnification power of 10× or less. Inother aspects, the cells are collected from the biological sample vialow cost filters and/or the biological sample is the obtained by manualdraw from the subject or low cost pump. In further aspects, colorimetricstaining is used to visualize the CAMLs and/or CTCs.

The invention is further directed to methods for using CAMLs and/or CTCsas companion diagnostics. Companion diagnostics is a useful tool inmatching drugs for specific treatment by evaluating the staining ofmarkers of the drug targets, evaluating gene amplifications ortranslocations by FISH, and using other molecular assays for specificgene mutations associated with the drugs, etc. The cells can serve inplace of tissue biopsies in determining whether certain therapies mightbe effective in the treatment of a subject having a disease, such ascancer. For example, the cells can be used in screening ofimmunotherapeutics to determine whether a cancer expresses the proteinrecognized by a given immunotherapeutic (e.g., an antibody). The cellscan also be assayed to determine whether the cells express certainpolynucleotides or whether the selected mutations are found in cellularDNA. As noted herein, CAMLs and CTCs often express or possess the samecancer markers as the cancer from which they are derived. Thus, and inan eighth embodiment, the invention is directed to a companiondiagnostic method for screening selected drug target markers in CAMLsand/or CTCs comprising collecting CAMLs and/or CTCs from a biologicalsample from a subject and determining whether the CAMLs and/or CTCsexpress or possess a selected drug target marker. In certain aspects,the drug target marker is cell surface marker, and the determining maybe, for example, via staining for the marker. As an example, PD-L1 canbe used as a cell surface marker for immunotherapeutics. In otheraspects, the drug target marker is a polynucleotide. In further aspects,the drug target marker is a genetic mutation, amplification ortranslocation and the determining may be, for example, via FISH.

Traditional methods of detection of viruses in blood are based on thepresence of antibodies or virally-infected cells. As CAMLs are a type ofimmune cell, they may also be used in a diagnostic for detection of aspectrum of viral infections, such as Human immunodeficiency virus(HIV), Hepatitis B virus (HBV), Epstein-Barr virus (EBV), and many more.Indeed, CAMLs have been found in blood of subjects with an active viralinfection. CAMLs engulf viral debris, cells infected by virus, orcellular debris that contains virus. One can stain for the viralmarker(s) directly in the CAMLs or perform molecular analysis of DNA orRNA in the CAML associated with the virus. Therefore, CAMLs can also beused as a biomarker to provide detection and diagnosis of active viralinfections, and in determining appropriate courses of therapy.

Furthermore, some viral infections such as HIV and HBV can lead tocancer. CAMLs found in the blood of those patients can be caused eitherby viral infection or by cancer itself. Staining for the cancer markeror for viral marker(s) can be used to provide diagnostic information.This may also be a useful method for early detection of virus-causedcancer.

Thus, and in a ninth embodiment, the invention is directed to methodsfor diagnosing a viral infection in a subject, comprising collectingCAMLs from a biological sample obtained from a subject and screening thecollected CAMLs for a virus. In certain aspects, the screening is viastaining CAMLs for a viral marker. The same cells can also bere-staining for additional viral markers. In further aspects, thescreening is via molecular analysis of DNA or RNA from the CAML. Inparticular aspects of this embodiment, a treatment decision is madebased on the outcome of the method. In particular aspects of thisembodiment, the method further comprises administering an anti-viraltherapeutic to the subject when a viral infection is diagnosed.

In an additional embodiment, the present invention includes methods ofmolecular analysis of a CAML comprising obtaining a single CAML cell andconducting molecular analysis on the single cell. There is no limitationon the particular type of molecular analysis that may be conducted onthe single cell and such means include, but are not limited to, nucleicacid sequencing, northern blot analysis and southern blot analysis.

In the relevant aspects and embodiments of the invention, the biologicalsample is one or more selected from the group consisting of peripheralblood, blood, lymph nodes, bone marrow, cerebral spinal fluid, tissue,and urine. The sample may be a fresh sample or a cryo-preserved samplethat is thawed. In a preferred aspect, the biological sample isperipheral blood. In other aspects, the blood is antecubital-vein blood,inferior-vena-cava blood or jugular-vein blood.

In the relevant aspects and embodiments of the invention, the cancer isone or more of a solid tumor, Stage I cancer, Stage II cancer, Stage IIIcancer, Stage IV cancer, carcinoma, sarcoma, neuroblastoma, melanoma,epithelial cell cancer, breast cancer, prostate cancer, lung cancer,pancreatic cancer, colorectal cancer, and other solid tumor cancers.

In the relevant aspects and embodiments of the invention, theanti-cancer therapeutic may be one or more of chemotherapy, radiationtherapy, immunotherapy, vaccine therapy, targeted therapy, and/or acombination of therapies.

In the relevant aspects and embodiments of the invention, CAMLs aredetected and/or collected using one or more means selected from thegroup consisting of size exclusion methodology, immunocapture, red bloodcell lysis, white blood cell depletion, FICOLL® (a hydrophilicpolysaccharide), electrophoresis, dielectrophoresis, flow cytometry andmicrofluidic chip, or a combination thereof. In a particular aspect, thesize exclusion methodology comprises use of a microfilter. Suitablemicrofilters can have a variety of pore sizes and shapes. When CAMLsalone are being detected and/or collected, the pore sizes can range fromabout 15 microns to about 20 microns. When both CAMLs and CTCs are beingdetected and/or collected, the pore sizes can range from about 5 micronsto about 10 microns. The larger pore sizes will eliminate most of theWBC contamination on the filter. The pores may have a round, race-trackshaped, oval, square and rectangular pore shape. In a preferred aspect,the microfilter has precision pore geometry and uniform poredistribution. In a particular aspect, CAMLs are detected and/orcollected using a microfluidic chip based on physical size-basedsorting, hydrodynamic size-based sorting, grouping, trapping,immunocapture, concentrating large cells, or eliminating small cellsbased on size. In a particular aspect, the CAMLs are detected and/orcollected using a CellSieve™ low-pressure microfiltration assay.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the present invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings, wherein:

FIGS. 1A-1I show a gallery of circulating cancer associatedmacrophage-like cells found in the blood of cancer patients. The mergedcolor images are generated by DAPI (blue), CK 8, 18 & 19 (green), EpCAM(red) and CD45 (violet).

FIGS. 2A-2F show a CAML with engulfed DNA fragment in the tail.

FIGS. 3A-3F show a CAML in the process of engulfing DNA fragment.

FIGS. 4A-4F show a CAML in the process of engulfing cytoplasm fragment.

FIGS. 5A-5F show a CAML with four engulfed white blood cells.

FIGS. 6A-6F show a CAML with two engulfed white blood cells.

FIGS. 7A-7F show a CAML in division.

FIGS. 8A-8F show two CAMLs after division.

FIGS. 9A-9F show two CAMLs after division.

FIGS. 10A-10F show a CAML with two small arms.

FIGS. 11A-11F show a CAML with two legs on the same side.

FIGS. 12A-12F show a CAML with two legs on the same side.

FIGS. 13A-13F show a CAML with very thin legs and rather regularnucleus.

FIGS. 14A-14F show a CAML isolated from a subject having a viralinfection.

FIGS. 15A-15F show a WBC bound to a CTC from a breast cancer patient.

FIGS. 16A-16F show a WBC bound to a CTC from a breast cancer patient.

FIGS. 17A-17F show a WBC bound to a CTC from a breast cancer patient.

FIGS. 18A-18F show a WBC bound to a CTC from a breast cancer patient.

FIGS. 19A-19F show a WBC bound to a CTC from a bladder cancer patient.

FIGS. 20A-20F show a WBC bound to a CTC from a bladder cancer patient.

FIGS. 21A-21F show a WBC bound to a CTC from a bladder cancer patient.

FIGS. 22A-22F show a WBC bound to a CTC from a kidney cancer patient.

FIGS. 23A-23F show a WBC bound to a CTC from a kidney cancer patient.

FIGS. 24A-24F show a WBC bound to a CTC from a kidney cancer patient.

FIGS. 25A-25F show a WBC bound to a CTC from a kidney cancer patient.

FIGS. 26A-26F show a WBC bound to a CTC from a kidney cancer patient.

FIGS. 27A-27F show a WBC bound to a CTC from a kidney cancer patient.

FIGS. 28A-28F show a WBC bound to a CTC from a kidney cancer patient.

FIG. 29 shows the frequency of CTC and CAMLs in 105 breast, prostate,pancreatic, and lung cancer patients and 30 health controls.

FIG. 30 shows the frequency of CAMLs in different stages of cancer from105 breast, prostate, pancreatic and lung cancer patients.

FIG. 31 shows the number of CAMLs following treatment.

FIG. 32 shows the number of CAMLs pre-surgical clinical assessment.

FIGS. 33A-33B shows the number of CAMLs based on pathological evaluationwith FIG. 33A showing the number for pathological confirmation and FIG.33B showing the cell size variation for the four different stages ofcancer.

FIG. 34 shows the re-staining of CAMLs.

FIG. 35 shows the effect of radiation therapy on cancer marker RAD50 ona CAML.

FIG. 36 shows the effect of radiation therapy on cancer maker RAD50, andPD-L1 on CTCs.

FIG. 37 shows the decrease of number of CAMLs after cancer treatmentover time.

FIG. 38 shows the decrease of size of CAMLs over time.

FIGS. 39A-39E show a CAML with strong stain for PD-L1 with Vimentin(max. S-N=880); PDL1 (max. S-N=880); CD45 (max. S-N=850); Length 107 μm.

FIGS. 40A-40E show a CAML with weak stain for PD-L1 with Vimentin (max.S-N=500); PDL1 (max. S-N=280); CD45 (max. S-N=820); Lengths 62 μm.

FIGS. 41A-41E show a CAML with very weak stain for PD-L1 with Vimentin(max. S-N=75); PDL1 (max. S-N=100); CD45 weak (max. S-N=145) Bright PDL1spot is 1000; CD45 is not smooth; Length—74 μm.

FIGS. 42A-42D show colorimetric staining of CAMLs.

DETAILED DESCRIPTION

The matters defined in the description such as a detailed constructionand elements are nothing but the ones provided to assist in acomprehensive understanding of the invention. Accordingly, those ofordinary skill in the art will recognize that various changes andmodifications of the embodiments described herein can be made withoutdeparting from the scope and spirit of the invention.

Cancer is the most feared illness in the world, affecting allpopulations and ethnicities in all countries. In the United Statesalone, there are more than 12 million cancer patients, with 1.7 millionnew cancer cases and almost 0.6 million deaths per year. Cancer deathworldwide is estimated to be about 8 million annually, of which 3million occur in developed countries where patients have availabletreatment.

Ideally there is a biomarker that can (i) provide early detection of allsolid tumors, especially for at risk groups such as smokers for lungcancer, (ii) confirm other indications of cancer, such as high PSA forprostate cancer, and/or (iii) provide early detection of cancerrecurrence.

Oncologists need to know how best to treat newly diagnosed cancerpatients. The current testing standard is a tissue biopsy, which is usedto determine the cancer subtype, because therapeutic drugs arefrequently effective only for specific subtypes. The biopsy methodvaries by location, but is invasive and can be risky.

To monitor treatment, oncologists need to know how well the drug isworking for the patient, whether the dose should be adjusted, andwhether the disease is spreading or responding to the drug. The commonmethods for answering these questions are x-ray computed tomography (CT)scans and magnetic resonance imaging (MRIs), both of which areexpensive. Additionally, these methods cannot provide the necessaryinformation until the tumor size has changed perceptibly.

Ninety percent of cancer patients die from metastasis, not from theprimary tumor. The metastatic process involves tumor cells that breakfree of the primary carcinomas (solid tumors of epithelial cells) andenter the blood stream. These breakaway cancer cells are known ascirculating tumor cells (CTCs). CTCs have the potential to be useful asa tool to determine therapy, monitor treatment, determine recurrence andprovide prognostic information of survival. However, CTCs cannot beconsistently collected from the blood even in stage III and IV cancers.

In this disclosure, a cell type is presented that is more consistentlyfound in the blood of solid tumor patients from stage I-IV. These cellsare macrophage-like cells that contain the same tumor markers as theprimary tumor and they are termed circulating Cancer AssociatedMacrophage-Like cells (CAMLs) herein.

CTCs and CAMLs can be found from the same patient sample at the sametime by size exclusion methods, such as by microfiltration methods.Microfilters can be formed with pores big enough to let all red bloodcells and majority of white blood cells through and retain larger cellssuch as CTCs and CAMLs. Size exclusion methods have also beenimplemented by microfluidic chips.

CAMLs have many clinical utilities when used alone. Furthermore, CAMLscan be combined with other markers such as CTCs, free DNA in blood andfree proteins in blood to further improve sensitivity and specificity ofa diagnosis. This is especially true for CAMLs and CTCs because they canbe isolated and identified at the same time.

Circulating Tumor Cells

The CTCs for many solid tumors express a number of cytokeratins (CKs).CK 8, 18, & 19 are the most commonly used in diagnostics, but surveyingneed not be limited to these markers. The surface of solid tumor CTCsusually express epithelial cell adhesion molecule (EpCAM). However, thisexpression is not uniform or consistent. CTCs should not express anyCD45, because it is a white blood cell marker. In assays to identifytumor associated cells, such as CTCs and CAMLs, it is sufficient to useantibody against markers associated with the solid tumor such as CK 8,18, & 19, or antibody against CD45 or DAPI. Combining the presence ofstaining with morphology, pathologically-definable CTCs (PDCTC),apoptotic CTCs and CAMLs can be identified (Adams, D. L., et al.,Cytometric characterization of Circulating Tumor Cells captured bymicrofiltration and their correlation to the CellSearch® CTC test.Cytometry Part A 2015; 87A:137-144).

PDCTCs for solid tumors express CK 8, 18, & 19, and can be identified bythe following characteristics:

-   -   A “cancer-like” nuclei stained by DAPI. The nuclei are usually        large with dot patterns. The exception is when the cell is in        division. The nucleus can also be condensed.    -   Expression of one or more of CK 8, 18 and 19; CTCs from        epithelial cancers usually express at least CK 8, 18 and 19. The        cytokeratins have a filamentous pattern.    -   Lack of CD45 expression.

An apoptotic CTC from a cancer that express CK 8, 18, & 19 is identifiedby the following characteristics:

-   -   A degrading nuclei.    -   Expression of one or more of CK 8, 18 and 19; the cytokeratins        are not filamentous in pattern, but appear fragmented in the        form of spots.    -   Lack of CD45 expression.

An apoptotic CTC of the present invention for solid tumors that expresscytokeratins thus includes those CTCs having one, two or three of thefollowing characteristics: (a) degrading nucleus; (b) expression of oneor more of cytokeratin 8, 18 and 19, and wherein the cytokeratin isfragmented in the form of spots; and (c) CD45 negative phenotype.

Detection of many carcinomas, sarcomas and melanomas can be through theidentification of a variety of other markers. For example, CTCs fromrenal cell cancer (RCC) and sarcomas express vimentin. CTC from bladdercancer usually express uroplakin and CK 8, 18 & 19 is weak. It ispossible to stain the cells for many different markers of interest.

CTCs of the present invention can also be characterized based on one ormore of the following characteristics: (i) number of CTCs; (ii) numberof WBCs bound to the CTCs; (iii) status of nucleus; (iv) degree ofcytokeratin 8 expression; (v) degree of cytokeratin 18 expression; (vi)degree of cytokeratin 19 expression; (vii) degree of EpCAM expression;(viii) degree of vimentin expression; (ix) degree of PD-L1 expression;(x) degree of uroplakin expression; (xi) cytokeratin morphology; (xii)location of markers (the location markers appear in CTCs, e.g.,cytoplasm versus nucleus, can change at the different time points); and(xiii) intensity of marker staining. The number of CTC characteristicsused in the methods of the invention can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 or all 13.

Circulating Cancer Associated Macrophage-like Cells (CAMLs)

CAMLs are characterized by having one or more of the following features:

-   -   CAMLs have a large atypical nucleus; multiple individual nuclei        can be found in CAMLs, though enlarged fused nucleoli are        common. CAML nuclei generally range in size from about 10 μm to        about 70 μm in diameter, more commonly from about 14 μm to about        64 μm in diameter.    -   For many cancers, CAMLs express the cancer marker of the        disease. For example, CAMLs associated with epithelial cancers        may express CK 8, 18 or 19, vimentin, etc. The markers are        typically diffused, or associated with vacuoles and/or ingested        material. The staining pattern for any marker is nearly        uniformly diffused throughout the whole cell. For sarcomas,        neuroblastomas and melanomas, other markers associated with the        cancers can be used instead of CK 8, 18, 19.    -   CAMLs can be CD45 positive.    -   CAMLs are large, approximately 20 micron to approximately 300        micron in size by diameter.    -   CAMLs are found in many distinct morphological shapes, including        spindle, tadpole, round, oblong, two legs, more than two legs,        thin legs, or amorphous shapes.    -   CAMLs typically have diffused cytokeratins.    -   If CAMLs express EpCAM, EpCAM is typically diffused throughout        the cell, or associated with vacuoles and/or ingested material,        and nearly uniform throughout the whole cell, but not all CAML        express EpCAM, because some tumors express very low or no EpCAM.    -   If CAMLs express a marker, the marker is typically diffused        throughout the cell, or associated with vacuoles and/or ingested        material, and nearly uniform throughout the whole cell, but not        all CAML express the same markers with equal intensity.    -   CAMLs express markers associated with the markers of the tumor        origin; e.g., if the tumor is of prostate cancer origin and        expresses PSMA, then CAML from this patient also expresses PSMA.        Another example, if the primary tumor is of pancreatic origin        and expresses PDX-1, then CAML from this patient also expresses        PDX-1. If the primary tumor or CTC of the cancer origin express        CXCR-4, then CAMLs from the patient also express CXCR-4.    -   If the primary tumor or CTC of the cancer origin expresses a        biomarker of a drug target, CAMLs express markers associated        with the markers of the drug target. An example of a biomarker        of immunotherapy is PD-L1.    -   CAMLs express monocytic markers (e.g. CD11c, CD14) and        endothelial markers (e.g. CD146, CD202b, CD31). CAMLs also have        the ability to bind Fc fragments.

CAMLs of the present invention thus includes those CAMLs having one,two, three, four or five of the following characteristics: (a) largeatypical nucleus having a size of about 14-64 μm; (b) expression of oneor more of cancer marker associated with the tumor, wherein the markeris diffused, or associated with vacuoles and/or ingested material; (c)cell size ranging from about 20 micron to about 300 microns; (d)morphological shape selected from the group consisting of spindle,tadpole, round, oblong, one or more legs, thin legs and amorphous; and(e) CD45 positive phenotype. CAMLs of the present invention also includethose CAMLs having one, two, three or four of the following additionalcharacteristics: (f) expression of diffuse EpCAM or vimentin with nearlyuniform distribution; (g) expression of one or more markers of a primarytumor; (h) expression of myeloid CD14 marker; (i) expression ofmonocytic CD11C markers; and (j) expression of endothelial CD146,CD202b, and CD31 markers. In a particular aspect, CAMLs of the presentinvention have each of the additional characteristics (f)-(j).

CAMLs of the present invention can also be characterized based on one ormore of the following characteristics: (i) number of CAMLs; (ii) averagesize of the CAMLs (CAML cell sizes range from about 20 micron to about300 microns in diameter); (iii) average size of the nuclei of the CAMLs(CAMLs have a large atypical nucleus having a size of about 14-64 μm indiameter); (iv) morphological shape of the CAMLs (CAML shapes includespindle, tadpole, round, oblong, two legs, more than two legs, thinlegs, or amorphous); (v) CD14 positive phenotype; (vi) degree of CD45expression; (vii) degree of EpCAM expression; (viii) degree of vimentinexpression; (ix) degree of PD-L1 expression; (x) degree of monocyticCD11C marker expression; (xi) degree of endothelial CD146 markerexpression; (xii) degree of endothelial CD202b marker expression; (xiii)degree of endothelial CD31 marker expression; (xiv) location of markers(the location markers appear in CAMLs, e.g., cytoplasm versus nucleus,can change at the different time points); (xv) presence of one or moremarkers associated with the cancer in the CAMLs, wherein the marker isdiffused, or associated with vacuoles and/or ingested material (e.g.,for epithelial cancer, the markers are cytokeratin 8, 18, and 19); and(xvi) intensity of marker staining. The number of CAML characteristicsused in the methods of the invention can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or all 16.

Under certain situations, it may be most suitable to stain CAMLs by H&Eor other colorimetric stains.

FIG. 1 contains a collage of CAMLs showing many of the different CAMLmorphologies and signal variation from separate prostate, breast andpancreatic patient samples (Adams, D., et al., Circulating giantmacrophages as a potential biomarker of solid tumors. PNAS 2014, 111(9):3514-3519): (FIG. 1A) pancreatic, (FIG. 1B) breast, (FIG. 1C)breast, (FIG. 1D) breast, (FIG. 1E) prostate, (FIG. 1F) pancreatic,(FIG. 1G) pancreatic, (FIG. 1H) prostate, and (FIG. 1I) prostate.Examples of morphology variants are as follows: amorphous (FIG. 1A),oblong (FIGS. 1B and 1G), spindle (FIGS. 1C, 1F, 1I, 3, 5, 6), round(FIG. 1D) and tadpole (FIGS. 1E & 1H). Color differences occur fromvarying degrees of protein expression from antibody reaction to EpCAM,cytokeratin and CD45.

FIGS. 2-13 show CAMLs stained with DAPI, CD10, vimentin and CD45, where(FIG. 2A) frames show merged microscope image DAPI (blue), CD10 (green),vimentin (red) and CD45 (violet), (FIG. 2B) frames show mergedmicroscope image DAPI (white), CD10 (green), vimentin (red) and CD45(violet), (FIG. 2C) frames show DAPI (white), (FIG. 2D) frames show CD10(white), (FIG. 2E) frames show vimentin (white), and (FIG. 2F) framesshow CD45 (white). Frame (FIG. 2B) with nucleus in white provides betterimage quality for some cells. Those cells possess the properties ofCAMLs described in paragraphs above. The choice of the stain was chosenbecause the source of the cells is kidney cancer patients.

FIGS. 2A-2F show engulfed DNA material at the end of top leg. FIGS.3A-3F show a CAML in the process of engulfing a cell, where the DNAmaterial is already in the CAML and some degraded cell cytoplasm isstill partially outside the CAML. FIGS. 4A-4F show a CAML in the processof engulfing degraded cellular material. This is most visible in thevimentin channel. FIGS. 5A-5F show a CAML with four engulfed CD45positive white blood cells. FIGS. 6A-6F show a CAML with two engulfedCD45 positive white blood cells. FIGS. 7A-7F appear to show a CAML inthe process of dividing. FIGS. 8A-8F show two similar side-by-side CAMLssuggesting the two cells might have come from the same origin. FIGS.9A-9F show another example of two similar side-by-side CAMLs.

FIGS. 10-12 show additional morphologies of CAML not identified inFIG. 1. FIGS. 10A-10F show a CAML with two small legs on the left sideof cell. FIGS. 11A-11F show a CAML with two legs on the same side. FIGS.12A-12F show a CAML with one leg on the right side and two legs on theleft side of the nucleus. FIGS. 13A-13F show a CAML with very thin legsand large single nucleus. FIGS. 14A-14F show a CAML found in patientwith HSV-2 viral infection.

Body's Immune Response to CTCs—T-Cells Bound to CTCs

When a tumor cell enters the blood stream, the CTC should be attacked byT-cells, resulting in the death of the tumor cell. When this happens,one or more T-cells will be bound to the CTC, resulting in the death ofthe CTC and eventual degradation of the CTC. T-cells are a subtype ofwhite blood cell. The CD45 marker stains WBCs and is not specific toT-cells. T-cells can be differentiated from granulocytes by themorphology of the nucleus. T-cells have a single nucleus approximatelyround and smaller than 8 microns. Filtration of the blood can capturewhite blood cells (WBCs) bound to the CTCs. The presence of WBCs boundto CTCs in the blood is an indication of presence of solid tumor andalso body's ability to eliminate the solid tumors. A determination onthe number of T-cells bound to CTCs can thus be used in diagnostics.

The markers of the tumor cell and the T-cell can be exchanged among thetwo cells when they come into contact. FIGS. 15-18 show WBCs bond toCTCs found in the blood of breast cancer patients. The markers used forthe breast cancer patients are DAPI, CK 8, 18 & 19, EpCAM and CD45.FIGS. 15-16 show the CTCs are apoptotic with CK 8, 18, and 19 degradedto spots. FIG. 18 shows a much degraded CTC losing both CK and EpCAMmarkers with no cytoplasm. It is often observed that the nuclei of theWBC and the CTCs are pulled towards each other as shown in FIG. 16.

FIGS. 19-21 shows WBCs bond to CTCs found in the blood of bladder cancerpatients. The markers used for the bladder cancer patients are DAPI, CK8, 18 & 19, EpCAM and CD45. In FIG. 19E, EpCAM is degraded to spots. Thecytoplasm of the WBC (marked in FIG. 19A) and the CTC are in the processof merging with EpCAM around the WBC. The WBC still expresses CD45.FIGS. 20A-20F show a still relatively intact CTC bound to a WBC. FIGS.21A-21F show a naked CTC nucleus without cytoplasm and the WBC (markedin FIG. 21A) still expressing CD45, but much weaker than WBCs not boundto the CTC (not marked in FIG. 21A).

FIGS. 22-28 show WBCs bond to a CTC found in the blood of kidney cancerpatients. The markers used for these patient samples are DAPI, CD10,vimentin and CD45. FIGS. 22A-22F show a CTC from mesenchymal kidneycancer with high expression of vimentin. It is tightly bound to the WBC.FIGS. 23-28 show WBCs bond to a CTC found in blood of non-mesenchymalkidney cancer patients expressing lower level of vimentin than shown inFIG. 22. The nuclei and the cytoplasm of the WBCs and CTCs are pulledtoward each other. CD10, vimentin and CD45 markers all become very weakafter WBCs bond to the CTCs. The amount of the cytoplasm decreases andeventually can all be lost.

Frequency of CTCs and CAMLs in Blood of Cancer Patients

PDCTCs are rarely found in early stages of cancer. Even though PDCTCsare found more frequently in stage III and IV breast and prostate cancerpatients, they can be seen at low frequencies in most other solidtumors. As an example, 105 cancer patients (breast (n=34), prostate(n=25), pancreatic (n=39) and lung (n=7)) and 30 health controls wereanalyzed. FIG. 29 shows PDCTCs and CAMLs could not be found in the bloodof healthy controls. In contrast, CAMLs were found in 98 out of 105cancer patients. The percentage of the patients with PDCTCs and CAMLsare 53% and 93%, respectively. The stage of cancer for the 105 cancerpatients were as follows: stage I (n=46), stage II (n=18), stage III(n=11), and stage IV (n=30). FIG. 30 shows that the percentage of thepatients having CAMLs in stages I, II, III and IV are 87%, 100%, 91% and97%, respectively. CAMLs were found to be more common than PDCTCs.Patient samples from 12 different solid tumors were analyzed: breast,prostate, pancreatic, lung, colorectal, uterine, neuroblastoma,esophageal, kidney, bladder, sarcoma, and ovarian. CAMLs were found inall those types of cancer (data not shown).

Number of CAMLs Vary Based on Therapy

Of the 105 patients noted above, 44 patients received no therapy, 12received target therapy and 49 received chemotherapy. Follow upscreening was performed to detect CAMLs in the patients after thecompletion of therapy. The number of CAMLs appears to depend on therapytype, as shown in FIG. 31. The number of CAMLs in patients receivingchemotherapy is much more than the number of CAMLs in patients receivingno therapy or targeted therapy.

Relationship of Number of CAMLs to Staging and Disease Progression

The number of CAMLs in patients undergoing chemotherapy is only weaklyassociated with cancer stage at the time of pre-treatment clinicalassessment, FIG. 32, and is highly correlative with stage afterpathological confirmation, FIG. 33A. For patients undergoingchemotherapy, the number of CAMLs were exponentially correlated withfinal pathological confirmation: stage I (3.2), Stage II (7.1), StageIII (14.6), Stage IV (35.1); R²=0.99.

FIG. 33B breaks down the number of CAMLs based on size for differentstages. CAMLs in later stages have more CAMLs with larger sizes.

Breast Cancer Screening

Because CAMLs can be found in high percentages in all stages of solidtumors, CAMLs as a cancer screening marker was evaluated for breastcancer. A double-blind prospective study was conducted of 41 subjectswhere mammography was judged abnormal. A double blinded test wasperformed: (i) 7.5 mL of peripheral blood samples were taken to test forCAMLs and (ii) tissue diagnosis by core needle biopsy was performed.Though mammography could not distinguish the subpopulations in thisgroup, CAML presence did differentiate between benign and malignantbreast disease with a sensitivity of 90% and a specificity of 72% (datanot shown).

Antibody Staining and Re-Staining of Isolated Cells

Typical fluorescent microscopes usually use four or five fluorescentchannels to minimize bleed through of fluorescent emissions intounintended fluorescent channels. One channel is taken by DAPI forimaging the nucleus. Often there is a need to evaluate more than threemarkers. Given these shortcomings, a method that allows analysis of upto approximately 12 different markers on the same cell was developedthat can be used in conjunction with each of the methods disclosed inthe present disclosure. After the process of filtration and staining thecells on the filter with a first set of markers, the cells of interestare identified and imaged. To evaluate more markers on the same cells, aquenching/stripping step was developed followed by a re-stainingtechnique. This required the cell to stay at the same location to allowreimaging of the same cell. This can be repeated a number of times. Toprow of FIG. 34 shows a CAML A with standard CTC stains: DAPI, CK 8, 18,19, EpCAM and CD45. The second row shows the re-staining of the samecells after quenching and re-stained for markers of interest: PD-L1, CCRand PD-1. Third row of FIG. 34 shows a CAML B with standard CTC stains.The fourth row shows the re-staining of the same CAML B after quenchingfor markers of interest: PD-L1, CCR and PD-1. This re-staining method isparticularly suitable for cells fixed on the microfilter. Their locationis fixed so they can be reimaged to evaluate different markers. CTCs andother cells on the filter can also be re-stained using this technique.This re-staining method is very useful for analyzing the cancer type,companion diagnostic, therapy response, cancer screening, and a varietyof research applications.

Single Cell Molecular Assays Using CAMLs and CTCs

Molecular analysis of CAMLs and CTCs can potentially be used todetermine cancer subtyping for gene mutations, translocations andamplifications, by various PCR assays, microarrays, FISH assays andsequencing. Single cell molecular analysis is becoming common, andsingle cell analysis of CAMLs are particularly interesting. Some assaysrequire more than one nucleus and/or cell to reduce errors. The presentinvention thus includes methods of molecular analysis of a single CAMLcell where a single CAML cell is obtained and molecular analysis isconducted on the single cell. There is no limitation on the particulartype of molecular analysis that may be conducted on the single cell andsuch means include, but are not limited to, nucleic acid sequencing,northern blot analysis and southern blot analysis.

A method to collect CTCs and CAMLs using the microfiltration device isdescribed. For this application, it is desired that the cells be easilyremoved from the filter, the opposite of the need for cells to stay onthe filter for re-staining purposes. The important step to allow removalof the cells is to coat the filter to prevent the cells from sticking,for example coating using fetal bovine serum (FBS) or bovine serumalbumin (BSA). Other coatings to prevent cell adhesion are alsoapplicable. The sample flows through the filter to collect cells largerthan the pores. There are two methods to collect the cells of interest.Method 1: remove the filter from the filter holder and place in a dishor glass slide with cells on top, and cover with appropriate liquid,such as PBS; cells can be directly picking cells off the filter usingmicromanipulators. Method 2: attach a syringe filled with PBS to thebottom of the holder with the cells on the filter and backwash the cellsoff the filter. The cells may be concentrated by centrifugation andremoving the supernatant. The cells need to be stained to enablevisualization under the microscope. One non-limiting choice of stains isfluorescent intercalating dyes. Another example is to stain for cellsurface markers such as EpCAM, CD45, and/or other markers. There arevarious ways to pick out cells of interest from the dish such asmicromanipulators, or instruments such as CellColector, and otherinstruments.

Companion Diagnostics

CAMLs can be used as a source of tissue for companion diagnostics todetermine the specific drug to be prescribed to the patient. Currentlycompanion diagnostics utilize tissue biopsies to stain for markers fordrug targets, perform FISH assays, and conduct other molecular assays tolook for gene mutations, amplification or translocations by PCR,microarrays, sequencing, etc. Examples of conventional companiondiagnostics utilizing tissue biopsies are FISH for HER2 amplification,FISH for ALK translocation, PD-L1 in tissue, AR and ER in tissue, etc.Sometime there is not enough tissue, or no tissue at all to evaluate awide variety of drugs. CTCs and CAMLs can be harvested repeatedly andused in place of the tissue biopsies. Also the same sample can bere-stained repeatedly to evaluate the efficacy of multiple drugs.

Monitor Treatment Response

Liquid Cell Biopsy provides a minimally invasive method to monitortreatment response in a patient. The following approaches can be adoptedto monitor efficacy of a cancer treatment, comprising:

-   (a) monitor the change of the number of CAMLs and CTCs from the same    subject at different time points after treatments;-   (b) monitor the changes of sizes of CAMLs and CTCs at the different    time points;-   (c) monitor the changes of intensity of markers in the CAMLs and    CTCs at the different time points; and-   (d) monitor the change of the location of the markers, cytoplasm    versus nucleus, in the CAMLs and CTCs at the different time points.

As one example related to chemotherapy, FIG. 31 shows that chemotherapyresponders seem to exhibit an increase in CAMLs shortly afterchemotherapy treatment. In contrast, the number of CAMLs from targettherapy did not show an increase above the no treatment control.

In a second example radiation therapy, the top row of FIG. 35 show aCAML from a lung cancer patient before radiation therapy stained forDAPI, PD-L1, RAD50 and PD-1. The bottom two rows show two differentCAMLs after radiation therapy also stained for DAPI, PD-L1, RAD50 andPD-1. RAD50 migrating to the site of DNA damage in the nucleus.

This change in RAD50 is also seen in CTCs. A third example combinesre-staining to examine effectiveness of radiation therapy. The top rowof FIG. 36 shows a cluster of CTCs from a lung cancer patient aftertreatment with radiation using the standard CTC stains: DAPI, CK 8, 18,19, EpCAM and CD45. The bottom row shows the same CTC cluster afterquenching and re-staining for markers related to radiation therapy andimmune response: PD-L1, RAD50 and PD-1.

A fourth example is related to immunotherapy. Immunotherapies thatenable the body to kill a tumor have shown impressive results for manytypes of cancers. An example of an immunotherapy drug is an antibodyagainst PD-L1 on the surface of the tumor cells; and another sample ofan immunotherapy drug is an antibody against PD-1 on the surface of thekiller T-cells. Both types of immunotherapy drug enable killer T-cellsto kill the tumor cells for some of the patients. FIG. 37 shows anexample of number of CAMLs collected at four time intervals spreadapproximately by two to three weeks. The treatment of PD-1 was on datesT1 and T3, after providing the blood sample for Liquid Cell Biopsy. Thedecrease in number of CAMLs after treatment may be an indication thatthe patient is not responding to treatment. FIG. 38 shows thecorresponding ranges of sizes of the CAMLs as the CAMLs are alsodecreasing in size. This information suggests that there might be adecrease of tumor debris in the blood as a result of treatment,suggesting that patient may not be responding to treatment. FIG. 39 is aCAML before T1 showing very bright PD-L1. The signal of PD-L1 abovebackground noise is about a factor of 8 of the background noise. FIG. 40is a CAML at T1 collected just before treatment. The PD-L1 signal is nowweak. The signal of PD-L1 above background noise is less than a factorof 2 of the background noise, indicating potential poor response. FIG.41 is a CAML at T3. The PD-L1 signal is now very weak. The signal ofPD-L1 above background noise is less than a factor of 1 of thebackground noise; this is an indication of loss of drug target, such asPD-L1.

A fifth example is related to monitoring the success of surgery. FigureS5 in paper by Adams et al. (Circulating giant macrophages as apotential biomarker of solid tumors. PNAS 2014, 111 (9):3514-3519)showed that surgery reduced the number of CAMLs. Continued presence ofCAMLs in the blood of the patient could indicate that cancer might notbe completely eradicated.

Capture of CAMLS and CTCs

Cells larger and/or less flexible than other cells present in a bodilyfluid may be collected by filtering the bodily fluid. For example,targeted cells indicative of a condition, e.g., CAMLs and CTCs, may becollected by passing a bodily fluid through a filter having openingsthat are too small for the target cells to pass through, but largeenough for other cells to pass through. Once collected, any number ofanalyses of the target cells may be performed. Such analyses mayinclude, for example, identifying, counting, characterizing expressionsof markers, obtaining molecular analysis, and/or culturing the collectedcells.

CAMLs, pathologically-definable CTCs, and apoptotic CTCs are larger thanred blood cells and most white blood cells. Using a precisionmicrofilter that has precision pore size and pore distribution has beenshown to provide high capture efficiency and low standard of deviationfor these cells. CellSieve™ microfilters (Creatv MicroTech) are oneexample of precision microfilters. CellSieve™ microfilters aretransparent and nonfluorescent making them ideal for microscope imaginganalysis. Pore sizes of 7-8 microns eliminated all the red blood cellsand 99.99% of the white blood cells. Methods to fabricate microfiltersproducing uniform pore size and distribution are described in WO2011/139445, and PCT/US12/66390, both of which is incorporated herein byreference in their entireties. Microfilters made by a track etch methodhave randomly located pores that can overlap resulting in effectivelylarge pores. They might lose some CAMLs and CTCs.

Besides microfiltration many other methods exist for capture of CTCs,and some can also be adopted to capture CAMLs. They generally break-downinto the following categories:

-   -   Because CAMLs are large compared with majority of blood cells,        many size based methods are suitable for capturing CAMLs.        Microfilters with 7-8 micron pores are ideal for simultaneously        capture of CAMLs and CTCs. If only CAMLs are of interest, not        CTCs, then the pore size can be larger to approximately 15-20        microns. The larger pore sizes will eliminate most of the WBC        contamination on the filter.    -   Immunocapture use ferrofluids, magnetic beads, microfluidic        chips, etc, coated with antibody for selection of CAMLs, or        elimination of other cells.    -   Red blood cell lysis can also be used for collecting CAMLs. The        resultant sample volume requires plating on multiple glass        slides.    -   White blood cell depletion.    -   FICOLL® (a hydrophilic polysaccharide).    -   Electrophoresis.    -   Dielectrophoresis.    -   Flow cytometry.    -   Microfluidic chips technologies that sort, select, group,        trapping, concentrates large cells or eliminate small cells by        size, utilization a variety of biological and physical        principles are also suitable.

Filtration is the best method to identify WBC bond to a CTC. Becauseboth the WBC and CTCs lose their markers and lose cytoplasm,immunocapture and flow cytometry are less suitable methods to isolatethem.

In the relevant aspects and embodiments of the invention, CTCs and WBCsbond to CTCs may be detected alone or in conjunction with the detectionof CAMLs. Such detection may be simultaneously or sequential detection,and can utilize the same or different means. For example, simultaneousdetection using a microfilter having a pore size that selects for bothcell types may be used. Suitable microfilters can have a variety of poresizes and shapes. Microfilters having pores of about 7-8 microns in sizeare acceptable, and include round, rectangular and race track poreshapes. Microfilters having round pores of about 7-8 microns in size areespecially optimal when polymeric microfilters are used. In a preferredaspect, the microfilter has precision pore geometry and uniform poredistribution.

CAML Isolation and Identification for Poor Economy

Methods for isolating CAMLs and/or CTCs from a biological sample andcounting the isolated cells using a camera, such as a cell phone camera,are embodiments of the present invention. Methods that utilize cameras,such as those on cell phones, may be used in those circumstances whereequipment and reagents required for detailed analysis of CAMLs and/orCTCs via marker staining and visualization are not available. Theability to count CAMLs and/or CTCs based on colorimetric staining may besufficient for some applications. When there is a lack of resources in acommunity, cancer tends to be diagnosed in late stages which translatesto limited treatment options and dim outcomes. A method to provide a lowcost diagnostic based on the counts of CAMLs and/or CTCs in a sample canadopt one or more of the following concepts.

-   -   (i) Utilize low cost filters with pore size ˜15-20 microns.    -   (ii) Filter the blood sample by manual draw or low cost pump.    -   (iii) Use colorimetric stain to visualize the CAMLs and/or CTCs.    -   (iv) Image cells using a cell phone camera with/without a        portable small lens, or a variety of white light microscope with        10× or lower magnification.

The large pore size in the filters will reduce the WBC contamination.Manual draw will reduce the cost. Colorimetric stain is low cost. Cellphone cameras can visualize CAMLs due to the large size of the cells.

Embodiments of Inventions

As suggested above, the unique characteristics of the CAMLs and CTCsdescribed herein make them well-suited for use in clinical methodologyincluding methods of screening and diagnosis diseases such as cancer,monitoring treatment, monitoring of disease progression and recurrence.

The invention is thus directed, in a first embodiment, to methods ofscreening a subject for cancer, comprising detecting CAMLs in abiological sample from a subject. In particular aspects, when CAMLs aredetected in the biological sample, the subject is identified aspotentially having a carcinoma, sarcoma, neuroblastoma, melanoma orother solid tumor. In other aspects, when CAMLs are detected in thebiological sample, the subject is identified as having a carcinoma,sarcoma, neuroblastoma, melanoma or other solid tumor. In certainaspects, the methods encompassed by this embodiment also includedetecting circulating tumor cells (CTCs) and T-cells bound to tumorcells in the biological sample. In particular aspects of this firstembodiment, the subject is a subject suspected of having cancer.

After CAMLs, CTCs or T-cells bound to tumor cells are found, it may bepossible to identify the type of tumor by staining, and in someinstances, re-staining these cells with markers associated with the typeof tumor. The National Cancer Institute tumor marker FactSheet listsmany cancer markers (see the NCI web site having the URL ending in“cancer.gov/cancertopics/factsheet/detection/tumor-markers” and“cancer.gov/about-cancer/diagnosis-staging/diagnosiskumor-markers-fact-sheet#q5”).Cancer markers are not limited to this list. A few examples of markerslisted below can be used to stain CAMLs and CTCs to provide initialindications of the cancer type:

-   -   BRAF mutation V600E: Cancer types: Cutaneous melanoma and        colorectal cancer    -   CA15-3/CA27.29: Cancer type: Breast cancer    -   CA19-9: Cancer types: Pancreatic cancer, gallbladder cancer,        bile duct cancer, and gastric cancer    -   CA-125: Cancer type: Ovarian cancer    -   Carcinoembryonic antigen (CEA): Cancer types: Colorectal cancer        and breast cancer    -   Cytokeratin fragments 21-1: Lung cancer    -   Estrogen receptor (ER)/progesterone receptor (PR): Cancer type:        Breast cancer    -   HE4: Cancer type: Ovarian cancer    -   HER2/neu: Cancer types: Breast cancer, gastric cancer, and        esophageal cancer    -   KIT: Cancer types: Gastrointestinal stromal tumor and mucosal        melanoma    -   Prostate-specific antigen (PSA) and PSMA: Cancer type: Prostate        cancer    -   Thyroglobulin: Cancer type: Thyroid cancer    -   5-Protein signature (Oval): Cancer type: ovarian cancer        The choice of markers is not limited to this list.

To identify one type of cancer, one marker may be sufficient for sometypes of cancer. To screen for more than one type of cancer in the sameblood sample, such as prostate, colorectal and lung cancers for man,re-staining of the CAMLs and CTCs with cancer markers specific for thosetypes of cancers is required after identifying the CAMLs and CTCs ofinterest. The following is an illustration of analyzing CAMLs and CTCsfor cancer screening up to four types of epithelial cancers usingmicrofiltration method:

-   -   Collect blood.    -   Isolate CTCs and CAMLs on the microfilter.    -   Stain the cells by DAPI, CK 8, 18, 19, CD14/CD45, and one marker        for one type of cancer.    -   Image the cells using fluorescent microscopes and identify CAMLs        and CTCs.    -   Quench the fluorescent dyes in the CAML and CTCs.    -   Re-stain the cells with DAPI and three additional markers of        interest.    -   Re-image the new markers in same CTCs and CAMLs previously        imaged.    -   Determine the type of cancer based on the markers.

CT scans for lung can show unusual findings as small as 4 mm in size. Itis now a recommended screening method for lung cancer. To verify that aninitial finding is lung cancer, tissue biopsy is needed. Tissue biopsyfor lung is very challenging and it is associated with higher risk ofcausing undesirable effects. Presence of CAMLs with associated lungcancer markers such as cytokeratin fragments 21-1, and other markers canbe used to provide a non-invasive step towards determine lung cancer.

For people carrying BRAC1 and BRAC2 mutations, they have a highprobability of getting breast and/or ovarian cancers. A blood test forCAMLs including the markers of CA125, Oval for ovarian cancer, and CEA,CA15-3/CA27.29, ER, PR and HER2 for breast cancer can be performed.

To screen top four types of cancer for man, one possible set of choicesfor markers can be PSMA for prostate cancer, CEA for colorectal cancer,cytokeratin fragments 21-1 for lung cancer and PDX-1 for pancreaticcancer.

The procedure and markers can vary depending on the CAML and CTCisolation method, the microscope, cancer types of interest, etc. Insummary, it is possible to screen for one specific cancer, a fewcancers, or any solid tumors in the category of carcinomas, sarcomas,neuroblastomas and melanomas. The markers do not need to be limited tothe ones describe here.

In a second embodiment, the invention is directed to methods fordiagnosing cancer in a subject, comprising detecting CAMLs in abiological sample from a subject, wherein when CAMLs are detected in thebiological sample, the subject is diagnosed with cancer. Theidentification of the specific type of cancer can be performed bystandard methods, such as staining for cancer markers. Theidentification of the specific type of cancer can also be performed bystaining for cancer markers and then re-staining the same cells foradditional markers. In certain aspects, the methods encompassed by thisembodiment also include detecting CTCs in the biological sample, whereinwhen CAMLs and CTCs are detected in the biological sample, the subjectis diagnosed with cancer. In certain aspects, the methods encompassed bythis embodiment also include detecting CTCs bound to a WBC and/ordetecting an apoptotic CTC bound to a WBC in the biological sample,wherein when CTCs bound to a WBC and/or an apoptotic CTC bound to a WBCare detected in the biological sample, the subject is diagnosed withcancer.

In a third embodiment, the invention is directed to methods fordetecting recurrence of cancer in a subject, comprising detecting CAMLsin a biological sample from a subject previously treated for cancer,wherein when CAMLs are detected in the biological sample, recurrence ofcancer is detected. The identification of the specific type of cancercan be performed by standard methods, such as staining for cancermarkers. The identification of the specific type of cancer can also beperformed by staining for cancer markers and then re-staining the samecells for additional markers. In certain aspects, the methodsencompassed by this embodiment also include detecting CTCs and/or WBCsbound to CTCs in the biological sample, wherein when CAMLs, CTCs andWBCs bound to CTCs are detected in the biological sample, recurrence ofcancer is detected. To identify the recurrence of the specific cancer,the stains should specifically include markers associated with thecancer in remission.

In a fourth embodiment, the invention is directed to methods forconfirming a diagnosis of cancer in a subject, comprising detectingCAMLs in a biological sample from a subject diagnosed with cancer,wherein when CAMLs are detected in the biological sample, a diagnosis ofcancer is confirmed in the subject. The invasive confirmation by tissuebiopsy may be avoided by most patients; the tissue biopsy would benecessary only when CAMLs are present. In certain aspects, the methodsencompassed by this embodiment also include detecting CTCs and/or WBCsbound to CTCs in the biological sample, wherein when one or more ofCAMLs, CTCs and WBCs bound to CTCs are detected in the biologicalsample, a diagnosis of cancer is confirmed in the subject. In particularaspects, the initial cancer diagnosis is via mammography, PSA test,presence of CA125, CT, MRI or PET imaging. In a particular aspect, thesubject is suspected of having cancer. The identification of thespecific type of cancer can be performed by standard methods, such asstaining for cancer markers. The identification of the specific type ofcancer can also be performed by staining for cancer markers and thenre-staining the same cells for additional markers.

In a fifth embodiment, the invention is directed to methods fordetermining cancer stage in a subject, comprising characterizing CAMLsin a biological sample from a subject having a cancer, wherein selectedcharacteristics of the CAMLs indicate cancer stage in the subject. Theidentification of the specific type of cancer can be performed bystandard methods, such as staining for cancer markers. Theidentification of the specific type of cancer can also be performed bystaining for cancer markers and then re-staining the same cells foradditional markers. In certain aspects, the methods encompassed by thisembodiment also include characterizing CTCs in the biological sample,wherein selected characteristics of the CAMLs and CTCs indicate cancerstage in the subject. In certain aspects, the CAMLs and/or CTCs arecollected from the biological sample prior to characterization. Thenumber of CAMLs in stage III and IV cancer are typically 5 or more in asample of peripheral blood in a volume of 7.5 ml. The percentage ofCAMLs larger than 40 microns in size by diameter is about 70% for StageIII and about 80% for Stage IV patients.

In a sixth embodiment, the invention is directed to methods formonitoring efficacy of a cancer treatment, comprising (a) assaying oneor more selected characteristics of CAMLs in a biological sample from asubject undergoing cancer treatment, and (b) comparing assay values forthe one or more selected characteristics determined in (a) to assayvalues for the same characteristics assayed in a similar biologicalsample from the same subject at one or more time points before, duringor after completion of treatment, wherein a change in one or more assayvalues indicates efficacy of the cancer treatment in the subject. Incertain aspects, the methods encompassed by this embodiment also include(a) assaying one or more selected characteristics of CTCs in thebiological sample, and (b) comparing assay values for the one or moreselected characteristics determined in (a) to assay values for the samecharacteristics assayed in a similar biological sample from the samesubject at one or more time points before, during or after completion oftreatment. In certain aspects, the CAMLs and/or CTCs are collected fromthe biological sample prior to characterization.

In certain aspects, the selected characteristics of the CAMLs are one ormore of: (i) change of the number of CAMLs from the same subject atdifferent time points after treatments; (ii) change in average size ofCAMLs at the different time points; (iii) change in intensity of markersin the CAMLs at the different time points; and (iv) change of locationof markers from nucleus to cytoplasm or vice versa in the CAMLs.

In certain aspects, the selected characteristics of the CTCs are one ormore of: (i) change of the number of CTCs in the biological samples atdifferent time points after treatment; (ii) change in intensity ofmarkers in the CTCs at the different time points; (iii) change oflocation of markers from nucleus to cytoplasm or vice versa; (iv) changein number of WBCs bound to CTCs in the biological sample; and (v) changein number of WBCs bound to CTCs in the biological samples at differenttime points after treatments.

The skilled artisan will understand that a change in the number of CAMLsand/or CTCs and/or WBCs bound to CTCs will be an indication of treatmentefficacy, where the change may be an increase or a decrease in thenumber of CAMLs and/or CTCs and/or WBCs bound to CTCs. The informationgathered on CAMLs, CTCs and CTC bound to WBC can be used independent ofeach other. The information gathered on CAMLs, CTCs and CTC bound to WBCcan also be used together.

The skilled artisan will understand that a change in the size of CAMLsand/or CTCs can be an indication of treatment efficacy, where the changein the size may be an increase or a decrease in size of CAMLs and/orCTCs and/or WBCs bound to CTCs. The information gathered on CAMLs andCTCs can be used independently or together.

The capability of tracking CAMLs provides a novel opportunity toroutinely monitor necrosis and chemotherapy or radiation therapyresponse. If the chemotherapy is not working, the CAMLs number will notincrease. This can be used in parallel with CTC detection. If thetreatment is working, the number of pathologically-definable CTCs willdecrease and number of apoptotic CTCs will increase. However, CTCscannot always be detected. If CTCs are detected at the same time asCAMLs, the sensitivity and specificity can be improved. For many cancersthere are large array of chemotherapy agents. If the patient is notresponding to one type of chemotherapy, the patient can quickly switchto another.

The invention is also directed to methods for isolating CAMLs and/orCTCs from a biological sample and counting the isolated cells using acamera, such as a cell phone camera, or white light microscope, or acamera attached to a white light microscope. Thus, and in a seventhembodiment, the present invention is directed to methods for detectingCAMLs and/or CTCs in a biological sample, comprising obtaining abiological sample from a subject, and detecting CAMLs and/or CTCs in thesample, wherein the detecting is via a camera, a white light microscopeor a camera attached to a white light microscope. In certain aspects,the camera is a cell phone camera. In certain aspects, the white lightmicroscope has a magnification power of 10× or less. In other aspects,the cells are collected from the biological sample via low cost filtersand/or the biological sample is the obtained by manual draw from thesubject or low cost pump. In further aspects, colorimetric staining isused to visualize the CAMLs and/or CTCs.

In an eighth embodiment, the invention is directed to a companiondiagnostic method for screening selected drug target markers in CAMLsand/or CTCs comprising collecting CAMLs and/or CTCs from a biologicalsample from a subject and determining whether the CAMLs and/or CTCsexpress or possess a selected drug target marker. In certain aspects,the drug target marker is cell surface marker, and the determining maybe, for example, via staining for the marker. As an example, PD-L1 canbe used as a cell surface marker for immunotherapeutics. In otheraspects, the drug target marker is a polynucleotide. In further aspects,the drug target marker is a genetic mutation, amplification ortranslocation and the determining may be, for example, via FISH.

In a ninth embodiment, the invention is directed to methods fordiagnosing a viral infection in a subject, comprising collecting CAMLsfrom a biological sample obtained from a subject and screening thecollected CAMLs for a virus. In certain aspects, the screening is viastaining CAMLs for a viral marker. In further aspects, the screening isvia molecular analysis of DNA or RNA from the CAML.

Traditional methods of detection of viruses are based on presence ofantibodies or viral particles in the blood. Because CAMLs engulf viraldebris, cells infected by virus, and cell debris that contains virus,the use of CAMLs in the manner can provide a useful tool in detectingand diagnosing viral infections. The source of the viral infection thatcan be diagnosed using these methods is not limited and includes, forexample Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV),Epstein-Barr Virus (EBV), among others.

Some viral infections, such as HIV and HBV, can lead to cancer. CAMLsfound in the blood of subjects having such infections can be causedeither by the viral infection or by cancer itself. Staining for cancermarkers or for viral markers can be used to provide diagnosticinformation. This may be a useful method for early detection ofvirus-caused cancers.

In the relevant aspects and embodiments of the invention, the therapiescomprise vaccination, chemotherapy, radiation therapy, immunotherapy,targeted therapy, and combinations thereof.

In the relevant aspects and embodiments of the invention, the biologicalsample may be any suspected of containing CTCs, WBCs bound to CTCs,and/or CAMLs. In certain aspects, the biological sample is one or moreselected from the group consisting of peripheral blood, blood, lymphnodes, bone marrow, cerebral spinal fluid, tissue, and urine. The samplemay be a fresh sample or a cryo-preserved sample that is thawed. In apreferred aspect, the biological sample is peripheral blood. In otheraspects, the blood is antecubital-vein blood, inferior-vena-cava bloodor jugular-vein blood.

Circulating monocytes have the ability to enter any tissue compartmentof the body, including lymph nodes, bone marrow, most organs, and evencross the blood brain barrier. The detection of CAMLs is therefore notlimited to blood, and the cells can also be found in lymph nodes, bonemarrow, cerebral spinal fluid, most organs, and urine.

In the relevant aspects and embodiments of the invention, the cancer isone or more of a solid tumor, Stage I cancer, Stage II cancer, Stage IIIcancer, Stage IV cancer, carcinoma, sarcoma, neuroblastoma, melanoma,epithelial cell cancer, breast cancer, prostate cancer, lung cancer,pancreatic cancer, colorectal cancer, and other solid tumor cancers. Theskilled artisan will appreciate that the methods of the invention arenot limited to particular forms or types of cancer and that they may bepracticed in association with a wide variety of cancers.

Since CAMLs can be found in stage I and II of cancer, CAMLs can be usedas screening for early detection of carcinomas, sarcomas, neuroblastomasand melanomas. Carcinomas are cancer of epithelial origin especially forhigh risk patients for breast, prostate, lung, pancreatic, colorectaland other cancers. Specificity of the type of cancer can be determinedby re-staining for various cancer site specific markers on the samecells captured on the microfilter. Some examples are (i) use antibodyagainst PSMA to specifically identifying prostate cancer, (ii) useantibody against PDX-1 to specifically identifying lung cancer, (iii)antibody against CA125 for ovarian cancer, and (iv) clorotoxin toidentify glioma.

Similarly CAMLs can be used to determine early recurrence of cancer whenthe cancer was under remission. Currently CT, MRI and PET imaging areused to monitor the patient's tumor, requiring the tumor to change insize substantially to notice the difference. Patients can therefore losevaluable time in beginning treatment when only subtle size changesoccur. CAMLs, alone or in combination with CTCs, can provide earlydetection of return of cancer. Non-invasive blood test of CAMLs and CTCsis much lower in cost than CT, MRI and PET imaging.

The CAMLs can also potentially be used to determine cancer subtyping orgene mutations, translocations or amplification. There are a number ofcancerous nuclei in each CAML. Thus, molecular analysis of the nucleusfor genetic mutation, genetic defects, and gene translocations canprovide information to determine treatments. There are drugs thatspecifically target certain gene mutations, translocation oramplifications. CAMLs can be used along or in parallel with CTCs formolecular analysis.

In the relevant aspects and embodiments of the invention, the volume ofthe biological sample will vary based on the source and/or identity ofthe sample. However, in the case of peripheral blood, the volume ofblood may range from about 0.5 ml to about 50 ml, about 1 ml to about 40ml, about 2 ml to about 30 ml, about 3 ml to about 25 ml, about 4 ml toabout 20 ml, about 5 ml to about 15 ml, about 6 ml to about 10 ml, orabout 7 ml to about 8 ml. A suitable volume also includes about 1, 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 ml.Blood volume typically used for detection of CTCs is 7.5 mL. Largervolumes of blood will provide more sensitivity and consistency, butsmaller volumes such as 3.5 mL may be sufficient. For many CTC detectionmethods, larger volumes of blood are not practical for a variety ofreasons. However, microfiltration of blood to capture CTCs and/or CAMLsallows more flexibility to increase the sample size. Blood volumes of 50mL have been shown to be successfully screened using CellSieve™microfilters with 160,000 pores. The suitable volume of blood to captureCAMLs would be 7.5 ml.

In the relevant aspects and embodiments of the invention, theanti-cancer therapeutic may be one or more of chemotherapy, radiationtherapy, immunotherapy, vaccine therapy, targeted therapy, and/or acombination of therapies.

In the relevant aspects and embodiments of the invention, CAMLs aredetected and/or collected using one or more means selected from thegroup consisting of size exclusion methodology, immunocapture, red bloodcell lysis, white blood cell depletion, FICOLL® (a hydrophilicpolysaccharide), electrophoresis, dielectrophoresis, flow cytometry andmicrofluidic chip, or a combination thereof. In a particular aspect, thesize exclusion methodology comprises use of a microfilter. Suitablemicrofilters can have a variety of pore sizes and shapes. When CAMLsalone are being detected and/or collected, the pore sizes can range fromabout 15 microns to about 20 microns. When both CAMLs and CTCs are beingdetected and/or collected, the pore sizes can range from about 5 micronsto about 10 microns, preferably 7-8 microns. The larger pore sizes willeliminate most of the WBC contamination on the filter. The pores mayhave a round, race-track shaped, oval, square and rectangular poreshape. In a preferred aspect, the microfilter has precision poregeometry and uniform pore distribution. In a particular aspect, CAMLsare detected and/or collected using a microfluidic chip based onphysical size-based sorting, hydrodynamic size-based sorting, grouping,trapping, immunocapture, concentrating large cells, or eliminating smallcells based on size. In a particular aspect, the CAMLs are detectedand/or collected using a CellSieve™ low-pressure microfiltration assay.

The results reported herein support the idea that CAMLs provide a robustindicator of cancer presence. The sensitivity and specificity of theutility of CAMLs can be further improved in combination withsimultaneous detection of CTCs and CTCs bond to WBCs. Cancer screeningis a strategy used in a population to identify an unrecognized diseasein individuals without signs or symptoms, with pre-symptomatic orunrecognized symptomatic disease. As such, screening tests are somewhatunique in that they are performed on persons apparently in good health.A screening test is not a diagnostic test. Diagnostic testing is aprocedure performed to confirm, or determine the presence of disease inan individual suspected of having the disease. CAMLs can be used as acancer diagnostic to provide additional non-invasive diagnostics toconfirm other screening techniques, such as mammography, PSA test,presence of CA125, CT, MRI or PET imaging.

We claim:
 1. A companion diagnostic method for screening selected drugtarget markers in Cancer Associated Macrophage-Like cells (CAMLs) and/orcirculating tumor cells (CTCs) comprising collecting CAMLs and/or CTCsfrom a biological sample from a subject and determining whether theCAMLs and/or CTCs express or possess a selected drug target marker. 2.The method of claim 1, wherein the selected drug target marker is a cellsurface marker.
 3. The method of claim 2, wherein the cell surfacemarker is PD-L1.
 4. The method of claim 1, wherein the selected drugtarget marker is a polynucleotide.
 5. The method of claim 1, wherein theselected drug target marker is a genetic mutation, amplification ortranslocation.